Significance of metal ions in galactose-1-phosphate uridylyltransferase: An essential structural zinc and a nonessential structural iron

Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). GalT is a metalloenzyme containing 1.2 mel of zinc and 0.7 mol of iron/mol of subunits [Ruzicka, F. J., Wedekind, J. E., Kim, J., Rayment, I., and Frey, P. A. (1995) Biochemistry 34, 5610-5617]. The zinc site lies 8 Angstrom from His 166 in active site, and the iron site lies 30 Angstrom from the active site [Wedekind,J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. Zinc is coordinated in tetrahedral geometry by Cys 52, Cys 55, His 115, and His 164. His 164 is part of the highly conserved active-site triad His 164-Pro 165-His 166, in which His 166 is the nucleophilic catalyst. Iron is coordinated in square pyramidal geometry with His 296, His 298, and Glu 182 in bidentate coordination providing the base ligands and His 281 providing the axial ligand. In the present study, site-directed mutagenesis, kinetic, and metal analysis studies show that: C52S-, C55S-, and HI64N-GalT are 3000-, 600-, and 10000-fold less active than wild-type. None of the variants formed the UMP-enzyme in detectable amounts upon reaction with UDP-Glc in the absence of Gal-1-P. Their zinc content was very low, and the zinc + iron content was about 50% of that for wild-type GalT. Mutation of His 115 to Asn 115 resulted in decreased activity to 2.9% of wild-type, with retention of zinc and iron. In contrast to the zinc-binding site, Glu 182 in the iron site is not important for enzymatic activity. The variant E182A-GalT displayed about half the activity of wild-type GalT, and all of the active sites underwent uridylylation to the UMP-enzyme, similar to wild-type GalT, upon reaction with UDP-Glc. Metal analysis showed that while E182A-GalT contained 0.9 equiv of zinc/subunit, it contained no iron. The residual zinc can be removed by dialysis with 1, 10-phenanthroline, with the loss in activity being proportional to the amount of residual zinc. It is concluded that the presence of zinc is essential for maintaining GalT function, whereas the presence of iron is not essential.